Flagellin gene, FlaC of Campylobacter

ABSTRACT

Purified and isolated nucleic acid molecules are provided which encode a FlaC flagellin protein of a strain of Campylobacter, particularly  C. jejuni,  or a fragment or an analog of the FlaC flagellin protein. The nucleic acid molecules may be used to produce proteins free of contaminants derived from bacteria normally containing the FlaA or FlaB proteins for purposes of diagnostics and medical treatment. Furthermore, the nucleic acid molecules, proteins encoded thereby and antibodies raised against the proteins, may be used in the diagnosis of infection.

REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.08/837,317 filed Apr. 11, 1997 (now U.S. Pat. No. 6,211,159).

FIELD OF THE INVENTION

The present invention is related to the molecular cloning of a geneencoding a flagellin protein, identified herein as FlaC, of theflagellar filament from a strain of Campylobacter.

BACKGROUND OF THE INVENTION

Campylobacter jejuni is a Gram-negative spiral microaerophilic bacteriumthat has been recognized as a cause of secretory type diarrhea andenteritis. (Ref. 1). Throughout this application, various references arereferred to in parenthesis to more fully describe the state of the artto which this invention pertains. Full bibliographic information foreach citation is found at the disclosed end of the specificationimmediately preceding the claims. These references are herebyincorporated by reference into the present disclosure). The flagellum ofC. jejuni is responsible for bacterial motility which enhances theorganism's pathogenicity. The flagellum consists of three majorcomponents; the filament, the hook, and the basal body (Ref. 2). Acampylobacter cell carries a single unsheathed flagellum at one or bothpoles of the body. The flagella are responsible for the high motility ofthe organisms as aflagellate mutants are nonmotile (Refs. 3, 4, 5, 6, 7,8, 9). A number of studies indicated that the polar flagellum plays animportant role in colonization of the viscous mucus lining of thegastric intestinal tract and that it is an important virulencedeterminant (Refs. 3,4,7,10,11).

The basic structure of the bacterial flagellum consists of a propeller(filament) connected via a universal joint (hook) to a transmissionshaft, motor and brushings (basal body) embedded in the cell envelope(Ref. 12). The flagellar filament consists of several thousandself-assembling protein (flagellin) monomers arranged in a helix. Theseform a hollow tube of relatively constant diameter and variable lengthwith an over corkscrew morphology.

Most eubacterial flagellar filaments that have been characterized appearto be composed of a single kind of flagellin (Ref. 8). However a numberof Eubacteria have now been shown to possess multiple flagellin genes(Refs. 6, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22), C jejuni (Refs. 13,15, 17, 21) and C. coli (Refs. 6, 13) have been reported to have twoflagellin genes (flaA and flaB). In C. jejuni, the flagellin genes flaAand flaB have been isolated and sequenced, however prior to the presentinvention a third flagellin gene had not been isolated andcharacterized.

It would be advantageous to provide nucleic acid molecules encodingflagellin proteins of the flagella for strains of Campylobacter andpurified flagellin proteins, including flaC for use as antigens,immunogenic compositions, including vaccines, carriers for otherantigens and immunogens and the generation of diagnostic reagents.

SUMMARY OF THE INVENTION

The present invention is directed towards the provision of purified andisolated nucleic acid molecules encoding a flagellin protein C (FlaC) ofa strain of Campylobacter or a fragment or an analog of the flagellinprotein. The nucleic acid molecules provided herein are useful for thespecific detection of strains of Campylobacter, and for diagnosis ofinfection by Campylobacter. The purified and isolated nucleic acidmolecules provided herein, such as DNA, are also useful for expressingthe flac gene by recombinant DNA means for providing, in an economicalmanner, purified and isolated FlaC proteins, subunits, fragments oranalogs thereof. The FlaC protein, subunits or fragments thereof oranalogs thereof, as well as nucleic acid molecules encoding the same andvectors containing such nucleic acid molecules, are useful inimmunogenic compositions against diseases caused by Campylobacter, thediagnosis of infection by Campylobacter and as tools for the generationof immunological reagents. Monoclonal antibodies or mono-specificantisera (antibodies) raised against the FlaC protein produced inaccordance with aspects of the present invention are useful for thediagnosis of infection by Campylobacter, the specific detection ofCampylobacter (in for example in vitro and in vivo assays) and for thetreatment of diseases caused by Campylobacter.

Peptides corresponding to portions of the FlaC protein or analogsthereof are useful immunogenic compositions against disease caused byCampylobacter, the diagnosis of infection by Campylobacter and as toolsfor the generation of immunological reagents. Monoclonal antibodies orantisera raised against these peptides, produced in accordance withaspects of the present invention, are useful for the diagnosis ofinfection by Campylobacter, the specific detection of Campylobacter (in,for example, in vitro and in vivo assays) and for use in passiveimmunization as a treatment of disease caused by Campylobacter.

In accordance with one aspect of the present invention, there isprovided a purified and isolated nucleic acid molecule encoding aflagellin protein (FlaC) of flagellum of a strain of Campylobacter, moreparticularly, a strain of Campylobacter jejunis, or a fragment or ananalog of the FlaC protein.

In one preferred embodiment of the invention, the nucleic acid moleculemay encode the FlaC protein of the Campylobacter strain.

In another aspect of the present invention, there is provided a purifiedand isolated nucleic acid molecule having a nucleotide sequence selectedfrom the group consisting of: (a) the entire nucleotide sequence set outin FIG. 1 (SEQ ID No: 1), or the complementary sequence of saidsequence; (b) the coding nucleotide sequence set out in FIG. 1 (SEQ IDNo: 2), or the complementary sequence of said sequence; (c) a nucleotidesequence encoding the amino acid sequence set forth in FIG. 1 (SEQ IDNo: 3); and (d) a nucleotide sequence which hybridizes under stringentconditions to any one of the sequences defined in (a), (b) or (c). TheDNA sequence defined in (c) preferably has at least about 90% sequenceidentity with any one of the DNA sequences defined in (a) and (b).

In an additional aspect, the present invention includes a vector adaptedfor transformation of a host, comprising a nucleic acid molecule asprovided herein. The vector may be one having the characteristics ofplasmid pD2-2.

The plasmids may be adapted for expression of the encoded FlaC protein,fragments or analogs thereof, in a heterologous or homologous host, ineither a lipidated or non-lipidated form. Accordingly, a further aspectof the present invention provides an expression vector adapted fortransformation of a host comprising a nucleic acid molecule as providedherein and expression means operatively coupled to the nucleic acidmolecule for expression by the host of the FlaC protein or the fragmentor analog of the FlaC protein. In specific embodiments of this aspect ofthe invention, the nucleic acid molecule may encode substantially allthe FlaC protein of the Campylobacter strain. The expression means mayinclude a nucleic acid portion encoding a leader sequence for secretionfrom the host of the FlaC protein or the fragment or the analog of theFlaC protein. The expression means also may include a nucleic acidportion encoding a lipidation signal for expression from the host of alipidated form of the FlaC protein or the fragment or the analog of theFlaC protein. The host may be selected from, for example, Escherichiacoli, Bordetella, Bacillus, Haemophilus, Moraxella, fungi, yeast orbaculovirus and Semliki Forest virus expression systems may be used.

In an additional aspect of the invention, there is provided atransformed host containing an expression vector as provided herein. Theinvention further includes a recombinant FlaC protein or fragment oranalog thereof producible by the transformed host. Further aspects ofthe present invention provide an isolated and purified FlaC protein of aCampylobacter strain substantially free from other proteins of theCampylobacter strain. The Campylobacter strain may be C. jejuni.

The present invention further provides synthetic peptides correspondingto portions of the FlaC protein. Accordingly, in a further aspect of theinvention, there is provided a synthetic peptide having no less than sixamino acids and no more than 150 amino acids and containing an aminoacid sequence corresponding to a portion only of a FlaC protein of astrain of Campylobacter or of a fragment or an analog of the FlaCprotein.

In accordance with another aspect of the invention, an immunogeniccomposition is provided which comprises at least one active componentselected from at least one nucleic acid molecule as provided herein, atleast one recombinant protein as provided herein, at least one of thepurified and isolated FlaC protein, as provided herein and at least onesynthetic peptide as provided herein, and a pharmaceutically acceptablecarrier therefor or vector therefor. The at least one active componentproduces an immune response when administered to a host.

The immunogenic compositions provided herein may be formulated as avaccine for in vivo administration to protect against diseases caused bybacterial pathogens that produce flagellin proteins. For such purpose,the compositions may be formulated as a microparticle, capsule, ISCOM orliposome preparation. Alternatively, the compositions may be provided incombination with a targeting molecule for delivery to specific cells ofthe immune system or to mucosal surfaces. The immunogenic compositionmay comprise a plurality of active components to provide protectionagainst disease caused by a plurality of species of flagellin proteinproducing bacteria.

The immunogenic compositions of the invention (including vaccines) mayfurther comprise at least one other immunogenic or immunostimulatingmaterial and the immunostimulating material may be at least one adjuvantor at least one cytokine. Suitable adjuvants for use in the presentinvention include (but are not limited to) aluminum phosphate, aluminumhydroxide, QS21, Quil A, derivatives and components thereof, ISCOMmatrix, calcium phosphate, calcium hydroxide, zinc hydroxide, aglycolipid analog, an octadecyl ester of an amino acid, a muramyldipeptide polyphosphazene, ISCOPREP, DC-chol, DDBA and a lipoprotein.Advantageous combinations of adjuvants are described in copending U.S.patent applications Ser. No. 08/261,194 filed Jun. 16, 1994 and Ser. No.08/483,856, filed Jun. 7, 1995, assigned to the assignee hereof and thedisclosures of which are incorporated herein by reference thereto.

In accordance with another aspect of the invention, there is provided amethod for inducing protection against infection or disease caused byCampylobacter or other bacteria that produce flagellin protein,comprising the step of administering to a susceptible host, which may bea primate, such as a human, an effective amount of the immunogeniccomposition as recited above.

In accordance with another aspect of the invention, an antiserum orantibody specific for the recombinant protein, the isolated and purifiedFlaC protein, synthetic peptide or the immunogenic composition, isprovided.

In a further aspect, there is provided a live vector for delivery ofFlaC protein to a host, comprising a vector containing the nucleic acidmolecule as described above. The vector may be selected from Salmonella,BCG, adenovirus, poxvirus, vaccinia and poliovirus. The nucleic acidmolecule may encode a fragment of the FlaC protein of a Campylobacterstrain which is conserved among bacteria that produce the FlaC protein.Such vector may be included in an immunogenic composition providedherein.

The present invention further includes a method of determining thepresence of nucleic acid encoding the FlaC protein of a strain ofCampylobacter, in a sample, comprising the steps of: (a) contacting thesample with the nucleic acid molecule provided herein to produceduplexes comprising the nucleic acid molecule and any said nucleic acidmolecule encoding the FlaC protein of Campylobacter present in thesample and specifically hybridizable therewith; and (b) determiningproduction of the duplexes.

In an additional aspect, the present invention provides a method ofdetermining the presence of a FlaC protein of a Campylobacter strain ina sample, comprising the steps of (a) immunizing a subject with theimmunogenic composition provided herein to produce antibodies specificfor the FlaC protein; (b) contacting the sample with the antibodies toproduce complexes comprising FlaC protein of a Campylobacter strainpresent in the sample and the FlaC protein specific antibodies; anddetermining production of the complexes.

A further aspect of the present invention provides a diagnostic kit fordetermining the presence of nucleic acid encoding the FlaC protein of astrain of Campylobacter, in a sample, comprising (a) the nucleic acidmolecule provided herein; (b) means for contacting the nucleic acid withthe sample to produce duplexes comprising the nucleic acid molecule andany said nucleic acid present in the sample and hybridizable with thenucleic acid molecule; and (c) means for determining production of theduplexes.

In another aspect of the present invention, there is provided adiagnostic kit for detecting the presence of a FlaC protein of aCampylobacter strain in a sample, comprising (a) a FlaC protein specificantibody to the immunogenic composition provided herein; (b) means forcontacting the antibody with the sample to produce a complex comprisingsaid FlaC protein and the antibody; and (c) means for determiningproduction of the complex.

The invention further includes the use of the nucleic acid molecules andproteins provided herein as medicines. The invention additionallyincludes the use of the nucleic acid molecules and proteins providedherein in the manufacture of medicaments for protection againstinfection by strains of Campylobacter.

The purified and isolated DNA molecules comprising at least a portioncoding for a FlaC protein of a species of Campylobacter typified by theembodiments described herein are advantageous as:

nucleic acid probes for the specific identification of Campylobacterstrains in vitro or in vivo.

the products encoded by the DNA molecules are useful as diagnosticreagents, antigens for the production of Campylobacter-specificantisera, for vaccination against the diseases caused by species ofCampylobacter and (for example) detecting infection by Campylobacter.

peptides corresponding to portions of the FlaC protein as typified bythe embodiments described herein are advantageous as diagnosticreagents, antigens for the production of Campylobacter-specificantisera, for vaccination against the diseases caused by species ofCampylobacter and (for example) for detecting infection byCampylobacter.

BRIEF DESCRIPTION OF DRAWINGS

The present invention will be further understood from the followingdescription with reference to the drawings in which:

FIG. 1 shows the nucleotide sequence of flaC gene and flanking regions(SEQ ID No: 1). The translated amino acid (SEQ ID No: 3) is locatedbelow the encoding sequence (SEQ ID No: 2). The ribosome binding site(RBS), the −10 and −35 regions are indicated by lines above thesequence. The putative transcriptional start sites are indicated by anasterisk below the sequence. Inverted repeats are shown with an arrowbelow the sequence.

FIG. 2 contains the restriction map of clone pD2-2 in pBluescriptvector. The location of the flaC is denoted by the boxed area below therestriction map. The restriction sites are : B, BglII; C, ClaI; H,HindIII; P, PvuI; R, RsaI; S,SalI; and X, XbaI. The shaded boxrepresents vector sequences. The direction of transcription is indicatedby the arrow.

FIG. 3 shows Southern Blot hybridization with flaC to HindIII digestedgenomic DNA from various Campylobacter species using 30% formamide. Lane1, 100 bp molecular marker; lane 2, blank; lane 3, C. jejuni ATCC 43431;lane 4, C. jejuni OH4382; lane 5, blank; lane 6, C. jejuni ATCC 43446;lane 7, C. jejuni ATCC 33559; lane 8, C. coli ATCC 43474; lane 9, C.lari ATCC 35221; lane 10, C. lari PC637; Lane 11, C. upsaliensis ATCC43954; lane 12, blank; lane 13, 100 bp molecular marker.

GENERAL DESCRIPTION OF THE INVENTION

Any Campylobacter strain may be conveniently used to provide thepurified and isolated nucleic acid, provided herein which may be in theform of DNA molecules, comprising at least a portion of the nucleic acidcoding for FlaC protein of a flagellum as typified by embodiments of thepresent invention. Such strains are generally available from clinicalsources and from bacterial culture collections, such as the AmericanType Culture Collection, Rockville, Md., U.S.A. One particular usefulspecies is C. jejuni.

In this application, the term “a flagellin protein C” is used to definea family of FlaC proteins which includes those having variations intheir amino acid sequences including those naturally occurring invarious strains of Campylobacter. The purified and isolated DNAmolecules comprising at least a portion coding for the FlaC protein ofthe present invention also include those encoding fragments orfunctional analogs of the FlaC protein. In this application, a firstprotein is a “functional analog” of a second protein if the firstprotein is immunologically related to and/or has the same function asthe second protein. The functional analog may be, for example, afragment of the protein or a substitution, addition or deletion mutantthereof.

Oligonucleotide probes derived from the Fur-box sequences from therecently cloned C. jejuni fur gene (Ref. 4) were used to screen apBluescript genomic library of C. jejuni. One of the clones sequenced,pD2-2 had an open reading frame (ORF) that codes for a polypeptide of249 amino acid residues. This protein had the highest homology with theN-terminal region of the C. coli FlaA protein sequence. The open readingframe was designated FlaC as the third flagellin gene in Campylobacter.

It is clearly apparent to one skilled in the art, that the variousembodiments of the present invention have many applications in thefields of vaccination, diagnosis, treatment of, for example,Campylobacter infections, and infections with other bacterial pathogensthat produce FlaC protein and the generation of immunological reagents.A further non-limiting discussion of such uses is further presentedbelow.

1. Vaccine Preparation and Use

Immunogenic compositions, suitable to be used as vaccines, may beprepared from FlaC proteins, analogs and fragments thereof, peptides andnucleic acid molecules encoding such FlaC proteins, fragments andanalogs thereof and peptides as disclosed herein. The vaccine elicits animmune response which produces antibodies, including anti-FlaC proteinantibodies and antibodies that are opsonizing or bactericidal. Shouldthe vaccinated subject be challenged by Campylobacter or other bacteriathat produce a FlaC protein, the antibodies bind to the basal body rodprotein and thereby inactivate the bacteria. Opsonizing or bactericidalantibodies may be particularly useful for providing protection.

Vaccines containing peptides are generally well known in the art, asexemplified by U.S. Pat. Nos. 4,601,903; 4,599,231; 4,599,230; and4,596,792; all of which references are incorporated herein by reference.Immunogenic compositions including vaccines may be prepared asinjectables, as liquid solutions or emulsions. The nucleic acidmolecules, FlaC protein, analogs and fragments thereof and/or peptidesmay be mixed with pharmaceutically acceptable excipients which arecompatible with the FlaC protein, fragments analogs or peptides. Suchexcipients may include, water, saline, dextrose, glycerol, ethanol, andcombinations thereof. The immunogenic compositions and vaccines mayfurther contain auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, or adjuvants to enhance the effectivenessof the vaccines. Immunogenic compositions and vaccines may beadministered parenterally, by injection subcutaneously orintramuscularly. Alternatively, the immunogenic compositions formedaccording to the present invention, may be formulated and delivered in amanner to evoke an immune response at mucosal surfaces. Thus, theimmunogenic composition may be administered to mucosal surfaces by, forexample, the nasal or oral (intragastric) routes. The immunogeniccomposition may be provided in combination with a targeting molecule fordelivery to specific cells of the immune system or to mucosal surfaces.Some such targeting molecules include vitamin B12 and fragments ofbacterial toxins, as described in WO 92/17167 (Biotech Australia Pty.Ltd.), and monoclonal antibodies, as described in U.S. Pat. No.5,194,254 (Barber et al). Alternatively, other modes of administrationincluding suppositories and oral formulations may be desirable. Forsuppositories, binders and carriers may include, for example,polyalkalene glycols or triglycerides. Oral formulations may includenormally employed incipients such as, for example, pharmaceutical gradesof saccharine, cellulose and magnesium carbonate. These compositionstake the form of solutions, suspensions, tablets, pills, capsules,sustained release formulations or powders and contain 1 to 95% of thenucleic acid molecule, FlaC protein, fragment analogs and/or peptides.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective,protective and immunogenic. The quantity to be administered depends onthe subject to be treated, including, for example, the capacity of theindividual's immune system to synthesize antibodies, and if needed, toproduce a cell-mediated immune response. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner. However, suitable dosage ranges are readily determinableby one skilled in the art and may be of the order of micrograms of theFlaC protein, analogs and fragments thereof and/or peptides. Suitableregimes for initial administration and booster doses are also variable,but may include an initial administration followed by subsequentadministrations. The dosage of the vaccine may also depend on the routeof administration and will vary according to the size of the host.

Thus, the nucleic acid molecules encoding the FlaC protein, fragments oranalogs thereof, of the present invention may also be used directly forimmunization by administration of the nucleic acid molecule (includingDNA molecules) directly, for example by injection for geneticimmunization or by constructing a live vector such as Salmonella, BCG,adenovirus, poxvirus, vaccinia or poliovirus. A discussion of some livevectors that have been used to carry heterologous antigens to the immunesystem are discussed in, for example, O'Hagan (Ref. 23). Processes forthe direct injection of DNA into test subjects for genetic immunizationare described in, for example, Ulmer et al. (Ref. 24).

The use of peptides in vivo may first require their chemicalmodification since the peptides themselves may not have a sufficientlylong serum and/or tissue half-life and/or sufficient immunogenicity.Such chemically modified peptides are referred to herein as “peptideanalogs”. The term “peptide analog” extends to any functional chemicalequivalent of a peptide characterized by its increased stability and/orefficacy and immunogenicity in vivo or in vitro in respect of thepractice of the invention. The term “peptide analog” is also used hereinto extend to any amino acid derivative of the peptides as describedherein. Peptide analogs contemplated herein are produced by proceduresthat include, but are not limited to, modifications to side chains,incorporation of unnatural amino acids and/or their derivatives duringpeptide synthesis and the use of cross-linkers and other methods whichimpose conformational constraint on the peptides or their analogs.

Examples of side chain modifications contemplated by the presentinvention include modification of amino groups such as by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; amidation with methylacetimidate; acetylation with aceticanhydride; carbamylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2, 4, 6, trinitrobenzenesulfonic acid (TNBS); alkylation of amino groups with succinic anhydrideand tetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5′-phosphate followed by reduction with NaBH₄.

The guanidino group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodimide activation viao-acylisourea formation followed by subsequent derivatisation, forexample, to a corresponding amide.

Sulfhydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of mixed disulphides with other thiol compounds;reaction with maleimide; maleic anhydride or other substitutedmaleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulfonic acid,phenylmercury chloride, 2-chloromercuric-4-nitrophenol and othermercurials; carbamylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides. Tryosine residuesmay be altered by nitration with tetranitromethane to form a3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives duringpeptide synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid-, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D-isomers of amino acids.

Immunogenicity can be significantly improved if the antigens areco-administered with adjuvants, commonly used as an 0.05 to 1.0 percentsolution in phosphate-buffered saline. Adjuvants enhance theimmunogenicity of an antigen but are not necessarily immunogenicthemselves. Adjuvants may act by retaining the antigen locally near thesite of administration to produce a depot effect facilitating a slow,sustained release of antigen to cells of the immune system. Adjuvantscan also attract cells of the immune system to an antigen depot andstimulate such cells to elicit immune responses.

Immunostimulatory agents or adjuvants have been used for many years toimprove the host immune responses to, for example, vaccines. Intrinsicadjuvants, such as lipopolysaccharides, normally are the components ofthe killed or attenuated bacteria used as vaccines. Extrinsic adjuvantsare immunomodulators which are typically non-covalently linked toantigens and are formulated to enhance the host immune responses. Thus,adjuvants have been identified that enhance the immune response toantigens delivered parenterally. Some of these adjuvants are toxic,however, and can cause undesirable side-effects, making them unsuitablefor use in humans and many animals. Indeed, only aluminum hydroxide andaluminim phosphate (collectively commonly referred to as alum) areroutinely used as adjuvants in human and veterinary vaccines. Theefficacy of alum in increasing antibody responses to diptheria andtetanus toxoids is will established and a HBsAg vaccine has beenadjuvanted with alum. While the usefulness of alum is well establishedfor some applications, it has limitations. For example, alum isineffective for influenza vaccination and inconsistently elicits a cellmediated immune response. The antibodies elicited by alum-adjuvantedantigens are mainly of the IgG1 isotype in the mouse, which may not beoptimal for protection by some vaccinal agents.

A wide range of extrinsic adjuvants can provoke potent immune responsesto antigens. These include saponins complexed to membrane proteinantigens (immune stimulating complexes ISCOMs), pluronic polymers withmineral oil, killed mycobacteria and mineral oil, Freund's completeadjuvant, bacterial products, such as muramyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes.

To efficiently induce humoral immune responses (HIR) and cell-mediatedimmunity (CMI), immunogens are emulsified in adjuvants. Many adjuvantsare toxic, inducing granulomas, acute and chronic inflammations(Freund's complete adjuvant, FCA), cytolysis (saponins and pluronicpolymers) and pyrogenicity, arthritis and anterior uveitis (LPS andMDP). Although FCA is an excellent adjuvant and widely used in research,it is not licensed for use in human or veterinary vaccines because ofits toxicity.

Desirable characteristics of ideal adjuvants include:

(1) lack of toxicity;

(2) ability to stimulate a long-lasting immune response;

(3) simplicity of manufacture and stability in long-term storage;

(4) ability to elicit both CMI and HIR to antigens administered byvarious routes, if required;

(5) synergy with other adjuvants;

(6) capability of selectively interacting with populations of antigenpresenting cells (APC);

(7) ability to specifically elicit appropriate T_(H)1 or T_(H)2cell-specific immune responses; and

(8) ability to selectively increase appropriate antibody isotype levels(for example, IgA) against antigens.

U.S. Pat. No. 4,855,283 granted to Lockhoff et al on Aug. 8, 1989 whichis incorporated herein by reference thereto teaches glycolipid analoguesincluding N-glycosylamides, N-glycosylureas and N-glycosylcarbamates,each of which is substituted in the sugar residue by an amino acid, asimmuno-modulators or adjuvants. Thus, Lockhoff et al. 1991 reported thatN-glycolipid analogs displaying structural similarities to thenaturally-occurring glycolipids, such as glycosphingolipids andglycoglycerolipids, are capable of eliciting strong immune responses inboth herpes simplex virus vaccine and pseudorabies virus vaccine. Someglycolipids have been synthesized from long chain-alkylamines and fattyacids that are linked directly with the sugars through the anomericcarbon atom, to mimic the functions of the naturally occurring lipidresidues.

U.S. Pat. No. 4,258,029 granted to Moloney and incorporated herein byreference thereto, teaches that octadecyl tyrosine hydrochloride (OTH)functions as an adjuvant when complexed with tetanus toxoid and formalininactivated type I, II and III poliomyelitis virus vaccine. Also,Nixon-George et al. (Ref. 25), reported that octadecyl esters ofaromatic amino acids complexed with a recombinant hepatitis B surfaceantigen, enhanced the host immune responses against hepatitis B virus.

Lipidation of synthetic peptides has also been used to increase theirimmunogenicity. Thus, Weismuller (Ref. 26), describes a peptide with asequence homologous to a foot-and-mouth disease viral protein coupled toan adjuvant tripalmityl-s-glyceryl-cysteinylserylserine, being asynthetic analogue of the N-terminal part of the lipoprotein from Gramnegative bacteria. Furthermore, Deres et al. (Ref. 27), reported in vivopriming of virus-specific cytotoxic T lymphocytes with syntheticlipopeptide vaccine which comprised of modified synthetic peptidesderived from influenza virus nucleoprotein by linkage to a lipopeptide,N-palmityl-s-[2,3-bis(palmitylxy)-(2RS)-propyl-[R]-cysteine (TPC).

2. Immunoassays

The FlaC protein, analogs and fragments thereof and/or peptides of thepresent invention are useful as immunogens, as antigens in immunoassaysincluding enzyme-linked immunosorbent assays (ELISA), RIAs and othernon-enzyme linked antibody binding assays or procedures known in the artfor the detection of anti-bacterial, Campylobacter, FlaC protein and/orpeptide antibodies. In ELISA assays, the FlaC proteins, analogs,fragments and/or peptides corresponding to portions of FlaC protein areimmobilized onto a selected surface, for example a surface capable ofbinding proteins or peptides such as the wells of a polystyrenemicrotiter plate. After washing to remove incompletely adsorbed FlaCprotein, analogs, fragments and/or peptides, a nonspecific protein suchas a solution of bovine serum albumin (BSA) or casein that is known tobe antigenically neutral with regard to the test sample may be bound tothe selected surface. This allows for blocking of nonspecific adsorptionsites on the immobilizing surface and thus reduces the background causedby nonspecific bindings of antisera onto the surface. The selectedpeptides may be from the conserved regions of FlaC protein to enhancethe cross-species detection unless one particular bacterial species isto be detected. In that event, a polypeptide is selected which is uniqueto the FlaC protein of that particular species. Normally, the peptidesare in the range of 12 residues and up and preferably 14 to 30 residues.It is understood however, that a mixture of peptides may be used eitheras an immunogen in a vaccine or as a diagnostic agent. There may becircumstances where a mixture of peptides from the conserved regionsand/or from the non-conserved regions are used to provide cross-speciesprotection and/or specific diagnosis. In this instance, the mixture ofpeptide immunogens is commonly referred to as a “cocktail” preparationfor use as a vaccine or diagnostic agent.

The immobilizing surface is then contacted with a sample such asclinical or biological materials to be tested in a manner conducive toimmune complex (antigen/antibody) formation. This may include dilutingthe sample with diluents such as BSA, bovine gamma globulin (BGG) and/orphosphate buffered saline (PBS)/Tween. The sample is then allowed toincubate for from 2 to 4 hours, at temperatures such as of the order of25° to 37° C. Following incubation, the sample-contacted surface iswashed to remove non-immunocomplexed material. The washing procedure mayinclude washing with a solution such as PBS/Tween, or a borate buffer.

Following formation of specific immunocomplexes between the test sampleand the bound FlaC protein, analogs, fragments and/or peptides, andsubsequent washing, the occurrence, and even amount, of immunocomplexformation may be determined by subjecting the immunocomplex to a secondantibody having specificity for the first antibody. If the test sampleis of human origin, the second antibody is an antibody havingspecificity for human immunoglobulins and in general IgG. To providedetecting means, the second antibody may have an associated activitysuch as an enzymatic activity that will generate, for example, a colordevelopment upon incubating with an appropriate chromogenic substrate.Quantification may then achieved by measuring the degree of colorgeneration using, for example, a visible spectra spectrophotometer.

3. Use of Sequences as Hybridization Probes

The nucleotide sequences of the present invention, comprising thesequence of the FlaC protein, fragments or analogs thereof, now allowfor the identification and cloning of the FlaC protein genes from anyspecies of Campylobacter and other bacteria that have FlaC proteingenes.

The nucleotide sequences comprising the sequence of the basal body rodprotein genes of the present invention are useful for their ability toselectively form duplex molecules with complementary stretches of otherFlaC protein genes. Depending on the application, a variety ofhybridization conditions may be employed to achieve varying degrees ofselectivity of the probe toward the other FlaC protein genes. For a highdegree of selectivity, relatively stringent conditions are used to formthe duplexes, such as low salt and/or high temperature conditions, suchas provided by 0.02 M to 0.15 M NaCl at temperatures of between about50° C. to 70° C. For some applications, less stringent hybridizationconditions are required such as 0.15 M to 0.9 M salt, at temperaturesranging from between about 20° C. to 55° C. Hybridization conditions canalso be rendered more stringent by the addition of increasing amounts offormamide, to destabilize the hybrid duplex. Thus, particularhybridization conditions can be readily manipulated, and will generallybe a method of choice depending on the desired results. In general,convenient hybridization temperatures in the presence of 50% formamideare: 42° C. for a probe which is 95 to 100% homologous to the targetfragment, 37° C. for 90 to 95% homology and 32° C. for 85 to 90%homology.

In a clinical diagnostic embodiment, the nucleic acid sequences of thebasal body rod protein genes of the present invention may be used incombination with an appropriate means, such as a label, for determininghybridization. A wide variety of appropriate indicator means are knownin the art, including radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of providing a detectable signal. Insome diagnostic embodiments, an enzyme tag such as urease, alkalinephosphatase or peroxidase, instead of a radioactive tag may be used. Inthe case of enzyme tags, calorimetric indicator substrates are knownwhich can be employed to provide a means visible to the human eye orspectrophotometrically, to identify specific hybridization with samplescontaining TfR gene sequences.

The nucleic acid sequences of FlaC protein genes of the presentinvention are useful as hybridization probes in solution hybridizationsand in embodiments employing solid-phase procedures. In embodimentsinvolving solid-phase procedures, the test DNA (or RNA) from samples,such as clinical samples, including exudates, body fluids or eventissues, is adsorbed or otherwise affixed to a selected matrix orsurface. The fixed, single-stranded nucleic acid is then subjected tospecific hybridization with selected probes comprising the nucleic acidsequences of the FlaC protein genes or fragments thereof of the presentinvention under desired conditions. The selected conditions will dependon the particular circumstances based on the particular criteriarequired depending on, for example, the G+C contents, type of targetnucleic acid, source of nucleic acid, size of hybridization probe etc.Following washing of the hybridization surface so as to removenon-specifically bound probe molecules, specific hybridization isdetected, or even quantified, by means of the label. As with theselection of peptides, it is preferred to select nucleic acid sequenceportions which are conserved among species of bacteria (includingCampylobacter) that produce FlaC proteins. The selected probe may be atleast 18 bp and may be in the range of 30 bp to 90 bp long.

4. Expression of the FlaC Flagellin Protein Genes

Plasmid vectors containing replicon and control sequences which arederived from species compatible with the host cell may be used for theexpression of the Flac protein genes in expression systems. The vectorordinarily carries a replication site, as well as marking sequenceswhich are capable of providing phenotypic selection in transformedcells. For example, E. coli may be transformed using pBR322 whichcontains genes for ampicillin and tetracycline resistance and thusprovides easy means for identifying transformed cells. The pBR322plasmid, or other microbial plasmid or phage must also contain, or bemodified to contain, promoters which can be used by the host cell forexpression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host can be used as a transforming vectorin connection with these hosts. For example, the phage in lambda GEM™-11may be utilized in making recombinant phage vectors which can be used totransform host cells, such as E. Coli LE392.

Promoters commonly used in recombinant DNA construction include theb-lactamase (penicillinase) and lactose promoter systems (Refs. 28, 29,30) and other microbial promoters such as the T7 promoter system (U.S.Pat. No. 4,952,496). Details concerning the nucleotide sequences ofpromoters are known, enabling a skilled worker to ligate themfunctionally with genes. The particular promoter used will generally bea matter of choice depending upon the desired results. Hosts that areappropriate for expression of the basal body rod protein genes,fragments, analogs or variants thereof include E. coli, Bacillusspecies, Campylobacter, fungi, yeast or the baculovirus expressionsystem may be used.

In accordance with this invention, it is preferred to make the proteinby recombinant methods, particularly when the naturally occurring FlaCprotein as purified from a culture of a species of Campylobacter mayinclude trace amounts of toxic materials or other contaminants. Thisproblem can be avoided by using recombinantly produced FlaC protein inheterologous systems which can be isolated from the host in a manner tominimize contaminants in the purified material. Particularly desirablehosts for expression in this regard include Gram positive bacteria whichdo not have LPS and are therefore endotoxin free. Such hosts includespecies of Bacillus and may be particularly useful for the production ofnon-pyrogenic basal body rod proteins, fragments or analogs thereof.

As noted above, bacteria that lack functional flagella and aresubstantially reduced in motility are also reduced in virulence. Thenucleic acid molecules encoding FlaC proteins of flagella as providedherein allow for the specific modification of flagella (by, for example,site-specific mutagenesis of the genes encoding the basal body proteins)to functionally disable the flagella. Bacteria having such functionallydisabled flagella will be rendered substantially non-motile andsubstantially avirulent. Such avirulent (or attenuated) bacteria areuseful as immunogens for vaccination against disease caused byCampylobacter or other bacteria that produced flagella containing FlaCproteins as encoded by genes of the present invention.

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitations.

Methods of molecular genetics, protein biochemistry, immunology andfermentation technology used but not explicitly described in thisdisclosure and these Examples are amply reported in the scientificliterature and are well within the ability of those skilled in the art.

Example 1

This Example describes the analysis of a clone encoding the FlaC proteinof the flagella of Campylobacter jejuni.

In ref. 4, are described the cloning and sequencing of the C. jejuni furgene and four Fur-box sequences were identified in the 5′ flankingregion. Oligonucleotides corresponding to the Fur-box sequences wereused as probes to screen a pBluescript genomic library of C. jejuni toattempt to isolate Fur-regulated genes.

One of the clones, plasmid pD2-2 (FIG. 2) contains the flaC gene of C.jejuni. The flaC gene in plasmid pD2-2 was sequenced by thedideoxy-chain termination method (reference 39 from paper) using theSequenase kit from United States Biomedical or by the cycle sequencingkit from Pharmacia. When necessary, synthetic oligonucleotide primerswere used in order to sequence of both strands. The determined nuclotidesequence (SEQ ID No: 1) is shown in FIG. 1.

A 16-mer oligonucleotide, 5′ ATTGCGCGAACAGCTG 3′, located on thecomplementary strand of the flaC gene at nt 314 to 298 was used tolocate the transcriptional start point of this gene. Threetranscriptional start points were detected at nt 183, nt 184, and nt185. Using the flaC encoding fragment as a probe, a single hybridizingRNA band of 2.0 kb was seen in a Northern blot analysis.

The flaC gene (SEQ ID No: 1) is a 747 nt sequence encoding a protein of249 amino acids (FIG. 1) (SEQ ID No: 3). The deduced molecular weight ofthis protein (FlaC) is 26.6 kDa. Nine nucleotides preceeding the Metstart codon is a good matched Shine-Dalgarno sequence AGAAGG. At nt167-174 is the −10 sequence (AATGATTA) and nt 145-148 is the −35sequence (TTAA) of the typical sigma 28 promoter sequences (FIG. 1). Thestop codon for the flaC gene is located at nucleotide 958.

Using pulsed field gel electrolysis and the flaC gene as a probe in aSouthern blot, the gene was mapped to the SalI-D fragment (ref. 31).FlgFG genes have been mapped to the same fragment and the flaC gene islocated less than 15 kb to the left (counterclockwise) from the basalrod genes (flgFG). The flaC gene is mapped on the opposite side of thegenome from where the flaAB gene cluster is located and are separated byabout 800 to 1000 kb.

Southern blot hybridization was performed in 30% formamide using a 0.5kb PvuII fragment from the coding region of flaC (FIG. 3). Thehybridization identified a single 0.8 kb HindIII band in all the C.jejuni strains tested, a 1.4 kb and a 1.1 kb band were detected in C.coli ATCC 33559 and ATCC 43474, respectively, a 1.8 kb band in C. lariATCC 35221 and PC637 as well as a 0.5 kb band in C. upsaliensis ATCC43954. These results suggest the pressure of a flaC homology among allthe Campylobacter species and strains which were tested.

Using high stringency hybridization conditions (50% formamide), only theband in the C. jejuni and C. coli strains was detected. For C. jejuniOH4382, the 0.8 kb band was faint, owing to insufficient DNA. Thepresence of the band was confirmed by a subsequent hybridization withthe flaC probe using a filter with equal amounts of genomic DNA from thethree C. jejuni strains. The 0.8 kb band was detected with equalintensity in all three strains.

SUMMARY OF THE DISCLOSURE

In summary of this disclosure, the flaC gene of Campylobacter jejuni hasbeen cloned and sequenced. The structural organization of the threeflagellin genes in C. jejuni is clearly different as flaC is mapped onthe opposite side of the genome from where the flaAB cluster islocated.Expression of the flaC gene can clearly be detected and a strongtranscriptional start site can be demonstrated. Modifications arepossible within the scope of this invention.

LIST OF REFERENCES

1. Penner J. L., (1988) Clin. Microbiol. Rev. 1: 157.

2. Macnab, Robert M. (1992) Annu. Rev. Genet. 26: 131.

3. Blaser, M. J. and L. B. Reller. (1981) New Engl. J. Med. 305:1444-1452

4. Chan, V. L. et al., (1995) Gene 164:25-31.

5. Grant, C. C. R. et al., (1993) Infect. Immun. 61:1764-1771.

6. Guerry, P. et al., (1991) J. Bacteriol. 173:4757-4764.

7. Morooka, T. A. et al., (1985) J.Gen. Microbiol. 131:1973-1980.

8. Wassenaar, T. M. et al., (1995) Can. J. Microbiol. 141:95-101.

9. Tao, R. et al., (1994) Mol. Microbiol. 14:883-893.

10. Newell, D. G. et al., (1985) J. Hyg. 95:217-227.

11. Pavlovskis, O. R. et al., (1991) Infect. Immun. 59:2259-2264.

12. Macnab, R. M. (1996) In Neidhardt F. C. et al., (ed.) 123-145.

13. Alm, R. A. et al., (1993) J. Bacteriol. 175:3051-3057.

14. Driks, A. R. et al., (1989) J. Mol. Biol. 206:627-636.

15. Fisher, S. H. and I. Nachamkin. (1991) Mol. Microbiol. 5:1151-1158.

16. Josenhans C. A. et al., (1995) J. Bacteriol. 177:3010-3020.

17. Khawaja, R. K. et al., (1992) Curr. Microbiol. 24:213-221.

18. Kostrzynska, M. et al., (1991) J. Bacteriol. 173:937-946.

19. McCarter, L. L. (1995) J. Bacteriol. 177:1595-1609.

20. Minnich, S. A. et al., (1988) J. Bacteriol. 170:3953-3960.

21. Nuijten, P. J. M. et al., (1990) J. Biol. Chem. 265:17798-17804.

22. Pleier, E. and R. Schmitt. (1991) J. Bacteriol. 173:2077-2085.

23. O'Hagan (1992) Clin Pharmokinet. 22:1.

24. Ulmer et al., (1993) Curr. Opinion Invest. Drugs. 2(9):983-989.

25. Nixon-George et al., (1990) J. Immunol. 14:4798.

26. Weismuller et al., (1989) Vaccine 8:29.

27. Deres et al., (1989) Nature 342:651.

28. Chang et al., (1978) Nature 375:615.

29. Itakura et al., (1977) Science 198:1056.

30. Goeddel et al., (1979) Nature 281:544.

31. Kim et al, J. Bacteriol. 175: 7468-7470

4 1 1105 DNA Campylobacter jejuni 1 atttgttttt tattttacta ataccataattgaactccaa aacttaggcg aaaacactac 60 aaaaactcaa gaatttatca gcatccataatgcaagtgaa gtggtgatta aaatcatctt 120 gataatgcaa gtttttttat atttcttaagctttaaaata gcaaaaaaat gataaaatat 180 taaataaatc caaaagagaa ggagtaggccatgatgatct ctgatgcaac tatgatgcaa 240 caaaattatt atttaaataa tgcacaaaaagctagcgata aagctttaga aaatattgca 300 gctgttcgcg caataagtgg agttgatagtgctaatttag ctattgctga ttctttaaga 360 tctcaatcaa gcactataga tcaaggtgtcgcaaatgctt atgatgctat aggggtttta 420 caaattgcag atgctagcct taccaatatctctcaaagcg cagatagact taatgaactt 480 tcagtaaaaa tgaacaatgc tgcacttaatgattctcaaa aaggaatgct aagaacagaa 540 gcaacacgca tacaagaatc catcaatgattcttttaata atgcaactta taatggaaaa 600 aatgtctttc aaactatgaa ttttgtagtaggtagcggaa ctgaaactac aaatttaaat 660 ccattagcaa cagatggatt aagcatagataatcaagata gtattacaaa ttttatggat 720 caacttggaa gtttaagaag tgaaataggctcaggtatca atgccatcac atcaaatatt 780 aatgcaagtg ttcaaaatag catcaactcaaaagcagctg aaaataattt actaaataat 840 gacatggcaa aaaatgtcaa tgattttaatgccaattatc taaaagaaaa tgctgctgct 900 tttgttgctg cgcaatccaa catgcagcttcaaagcaaaa ttgctaattt attacaataa 960 aataagccct aaatagggct tattttttatcaaaatgact ttagagcaaa ttttagaaaa 1020 aaccaaaaac gttcgtcttg tagcggcaagcaagtatgtc gatgcaagta taattgaaaa 1080 gctttttgat caaggtatag tagaa 1105 2747 DNA Campylobacter jejuni 2 atgatgatct ctgatgcaac tatgatgcaacaaaattatt atttaaataa tgcacaaaaa 60 gctagcgata aagctttaga aaatattgcagctgttcgcg caataagtgg agttgatagt 120 gctaatttag ctattgctga ttctttaagatctcaatcaa gcactataga tcaaggtgtc 180 gcaaatgctt atgatgctat aggggttttacaaattgcag atgctagcct taccaatatc 240 tctcaaagcg cagatagact taatgaactttcagtaaaaa tgaacaatgc tgcacttaat 300 gattctcaaa aaggaatgct aagaacagaagcaacacgca tacaagaatc catcaatgat 360 tcttttaata atgcaactta taatggaaaaaatgtctttc aaactatgaa ttttgtagta 420 ggtagcggaa ctgaaactac aaatttaaatccattagcaa cagatggatt aagcatagat 480 aatcaagata gtattacaaa ttttatggatcaacttggaa gtttaagaag tgaaataggc 540 tcaggtatca atgccatcac atcaaatattaatgcaagtg ttcaaaatag catcaactca 600 aaagcagctg aaaataattt actaaataatgacatggcaa aaaatgtcaa tgattttaat 660 gccaattatc taaaagaaaa tgctgctgcttttgttgctg cgcaatccaa catgcagctt 720 caaagcaaaa ttgctaattt attacaa 747 3249 PRT Campylobacter jejuni 3 Met Met Ile Ser Asp Ala Thr Met Met GlnGln Asn Tyr Tyr Leu Asn 1 5 10 15 Asn Ala Gln Lys Ala Ser Asp Lys AlaLeu Glu Asn Ile Ala Ala Val 20 25 30 Arg Ala Ile Ser Gly Val Asp Ser AlaAsn Leu Ala Ile Ala Asp Ser 35 40 45 Leu Arg Ser Gln Ser Ser Thr Ile AspGln Gly Val Ala Asn Ala Tyr 50 55 60 Asp Ala Ile Gly Val Leu Gln Ile AlaAsp Ala Ser Leu Thr Asn Ile 65 70 75 80 Ser Gln Ser Ala Asp Arg Leu AsnGlu Leu Ser Val Lys Met Asn Asn 85 90 95 Ala Ala Leu Asn Asp Ser Gln LysGly Met Leu Arg Thr Glu Ala Thr 100 105 110 Arg Ile Gln Glu Ser Ile AsnAsp Ser Phe Asn Asn Ala Thr Tyr Asn 115 120 125 Gly Lys Asn Val Phe GlnThr Met Asn Phe Val Val Gly Ser Gly Thr 130 135 140 Glu Thr Thr Asn LeuAsn Pro Leu Ala Thr Asp Gly Leu Ser Ile Asp 145 150 155 160 Asn Gln AspSer Ile Thr Asn Phe Met Asp Gln Leu Gly Ser Leu Arg 165 170 175 Ser GluIle Gly Ser Gly Ile Asn Ala Ile Thr Ser Asn Ile Asn Ala 180 185 190 SerVal Gln Asn Ser Ile Asn Ser Lys Ala Ala Glu Asn Asn Leu Leu 195 200 205Asn Asn Asp Met Ala Lys Asn Val Asn Asp Phe Asn Ala Asn Tyr Leu 210 215220 Lys Glu Asn Ala Ala Ala Phe Val Ala Ala Gln Ser Asn Met Gln Leu 225230 235 240 Gln Ser Lys Ile Ala Asn Leu Leu Gln 245 4 16 DNACampylobacter jejuni 4 attgcgcgaa cagctg 16

What we claim is:
 1. A recombinant FlaC protein of a strain ofCampylobacter producible by a transformed host cell containing anexpression vector adapted for transformation of the host cell comprisinga nucleic acid molecule which is selected from the group consisting of:(a) the entire nucleotide sequence set out in FIG. 1 (SEQ ID No: 1), orthe complementary sequence of said sequence; (b) the coding nucleotidesequence set out in FIG. 1 (SEQ ID No: 2), or the complementary sequenceof said sequence; (c) a nucleotide sequence encoding the amino acidsequence set out in FIG. 1 (SEQ ID No: 3); and (d) a nucleotide sequenceencoding a flagellin protein C (FlaC) of a flagellin of a strain ofCampylobacter and which has at least about 90% sequence identity withany one of the nucleotide sequences defined in (a), (b) or (c), andexpression means operatively coupled to the nucleic acid molecule forexpression by the host cell of the FlaC protein of a strain ofCampylobacter.
 2. An isolated and purified FlaC protein of aCampylobacter strain, free from other proteins of the Campylobacterstrain.
 3. The FlaC protein of claim 2 wherein said strain ofCampylobacter is a Campylobacter jejuni strain.
 4. An immunogeniccomposition, comprising at least one active component selected from thegroup consisting of: (A) a recombinant FlaC protein of a strain ofCampylobacter producible in a transformed host cell containing anexpression vector comprising a nucleic acid molecule which is selectedform the group consisting of: (a) the entire nucleotide sequence set outin FIG. 1 (SEQ ID No: 1), or the complementary sequence of saidsequence; (b) the coding nucleotide sequence set out in FIG. 1 (SEQ IDNo: 2), or the complementary sequence of said sequence; (c) a nucleotidesequence encoding the amino acid sequence set out in FIG. 1 (SEQ ID No:3); and (d) a nucleotide sequence encoding a flagellin protein C (FlaC)of a flagellin of a strain of Campylobacter and which has at least about90% sequence identity with any one of the nucleotide sequences definedin (a), (b) or (c), and expression means operatively coupled to thenucleic acid molecule for expression by the host cell of the FlaCprotein of a strain of Campylobacter or (B) an isolated and purifiedFlaC protein of a Campylobacter strain free from other proteins of theCampylobacter strain; and a pharmaceutically acceptable carriertherefor, said at least one active component producing an immuneresponse when administered to a host.